HCV-RNA和抗-HCV的联合检测在丙型肝炎确诊中的临床意义

目的:探讨抗-HCV和HCV-RNA的联合检测在丙型肝炎确诊中的临床意义。方法:采用ELISA法检测抗-HCV及实时荧光定量PCR检测HCV-RNA。结果:在急性丙型肝炎组中抗-HCV和HCV-RNA同时阳性所占百分率(29.8%)显著低于慢性丙型肝炎患者(62.3%)(P<0.05);抗-HCV阴性和HCV-RNA阳性在急性丙型肝炎组所占百分率(58.3%)显著高于慢性丙型肝炎患者(24.6%)(P<0.05);经相关性分析,ALT含量与HCV-RNA含量呈正相关性(r=0.725,P<0.05)。结论:临床抗-HCV和HCV-RNA的联合检测有助于丙型肝炎的早期明确诊断。     

丙型肝炎病毒不同标志物检测结果的相关性分析

目的分析丙型肝炎病毒(Hepatitis C virus,HCV)抗原、抗体及核酸标志物实验室检测结果之间的关联性,评价适合血源筛查与早期临床确诊联合检测HCV感染的实验室诊断技术。方法用HCV RNA定量试剂、HCV核心抗原试剂及HCV抗体试剂分别检测304份血浆样本中HCV RNA载量、HCV Ag和抗-HCV指标,并对HCV RNA阳性样本进行基因分型。结果在304份血浆样本中,检出HCV RNA阳性样本87份,其HCV RNA载量≥500 IU/m L、500~30 IU/m L之间和<30 IU/m L时,血清学标志物HCV Ag浓度≥3 fmol/L及抗-HCV信号值/判断值≥1的阳性率分别为92.0%(23/25):96.0%(24/25)、58.8%(10/17):82.3%(14/17)及11.1%(5/45):75.6%(34/45),HCV RNA载量低于基因分型试剂盒LOD要求为500 IU/m L时,基因分型检测率为24.2%(15/62)。HCV RNA阴性样本217份中,HCV Ag浓度≥3 fmol/L和抗-HCV信号值/判断值≥1的样本阳性率分别为3.2%(7/217)和32.7%(71/217)。血清学指标在不同HCV RNA载量的阳性率差异具有统计学意义(χ2=197.4,P<0.01),HCV RNA载量与HCV Ag和抗-HCV水平成正相关。结论在中国人群中存在低HCV RNA水平携带者,HCV Ag和基因分型检测能力需要进一步提高。     

血源肝炎病毒筛查试验结果的分析

目的 探讨血筛酶免疫试剂的质量以及2次化验试剂的合理搭配,最大限度地避免漏检和减少假阳性。方法 对随机抽取的73份HBsAg和99份抗-HCV检测不合格的献血标本分别采用与常规筛查相同的2种进口试剂和另1种国产酶免试剂进行复检,并对复检中至少1种试剂为反应性的标本进行HBsAg中和试验或抗-HCV重组免疫印迹试验确证。结果 73份HBsAg不合格标本中,20份确证阳性,11份可疑,42份阴性;99份抗-HCV不合格标本中,29份确证阳性,70份阴性。针对研究范围内特定的标本,进口试剂的漏检率可达13.79%～27.59%,且也存在不同程度的假阳性。2种进口试剂间检测结果互补,而国产试剂无法查出进口试剂的漏检。结论 无论是进口还是国产血筛酶免试剂,都存在不同程度的漏检和假阳性情况,应选择2种检测结果互补的试剂对血液进行筛查,以保障血液安全。     

268份人类免疫缺陷病毒抗体待确定样本的检测结果分析

为了提高实验室人类免疫缺陷病毒(human immunodeficiency virus,HIV)抗体检测能力及对免疫蛋白印迹(Western blot,WB)实验结果的判断能力,对送检至北京市东城区艾滋病确证实验室的268份HIV抗体待确定样本进行确证实验及结果分析。按照试剂说明书和实验室标准作业程序(Standard Operation Procedure,SOP)操作对送检的全部样本进行WB确证实验;了解HIV筛查实验结果与确证实验结果的相关性,并分析不同送检机构、送检人群样本的检测结果差异以及不同试剂、不同检测方法的结果差异。结果显示在筛查出抗体待确定的268份样本中,确证阳性170份,阳性率63.43%;确证阴性51份,阴性率19.03%;不确定结果47份,占筛查有反应的17.54%。确证阳性病例来自监管场所、自愿咨询检测门诊(Voluntary Counseling and Test,VCT)和医疗机构,不同送检单位及不同人群的阳性样本率有显著统计学意义(P<0.01)。WB确证阳性样本带型以全条带和次全条带为主,且所有确证阳性标本均来自双试剂阳性样本。不同检测方法阳性样本率的差异有显著统计学意义(P<0.01),其中化学发光法的样本阳性率占46.27%,酶联免疫吸附实验(ELISA)占88.29%,胶体硒法占43.48%。本研究结果提示,对潜在HIV感染者,应扩大检测面,加强医疗机构检测,并提供一种以上方法的多次检测,以减少漏检的风险。     

类风湿因子分型(IgG、IgM、IgA)与丙型肝炎关系的初探

为了了解类风湿因子(RF)自身免疫抗体的分型(IgG、IgM、IgA)与丙型肝炎之间的关系,探讨其出现的规律及与肝功能损坏情况的相关性.采用定量双抗原夹心ELISA法进行检测.结果发现抗-HCV阴性、ALT＜40U的C组RF总阳性率为5%;抗-HCV阳性、ALT＜40U的A组为56%:抗-HCV阳性,ALT≥40U的B组为89%,C组与A、B组之间有显著性的差异P＜0.01).A组lgM-RF阳性率为4%;B组为56%;A组与B组之间有显著性差异(P＜0.05).因此抗-HCV阳性者血清中各型RF总阳性率比正常对照的C组有明显的增高;B组的IgM-RF比A组明显地增高.研究结果提示:IgM-RF的增高与丙肝患者肝功能损坏的发生与否存在密切的关系.     

抗—HCV阴性献血员中丙型肝炎病毒RNA检测及序列分析

对95份抗—HCVIgG阴性献血员采用逆转录聚合酶链反应法（PCR）检测丙型肝炎病毒RNA,结果8次中有6次其检测出17份阳性标本（17／95,17．9％）,复查抗—HCVIgG仍为阴性。对其中8份阳性产物中高变区1的序列分析结果表明均为不同株HCV序列．排除了PCR污染的可能性。对其中2份阳性产物测定了全序列并与HCV各基因型代表株的相应序列比较,与HCVⅡ型相应序列的核苷酸同源性为77％～79％。而与HCVⅠ、Ⅲ、Ⅳ相应序列间的同源性为62％～69％,表明为HCVⅡ型序列。结果提示献血员抗—HCVIgG筛选不能完全排除HCV感染者,漏检不是由于HCV基因序列变异．而是检测方法本身缺陷所致。     

乙型肝炎血清标志模式与病毒载量的关系及意义

为探讨乙型肝炎 (以下简称乙肝 )血清标志模式与病毒载量的关系及临床意义 ,作者选择符合 2 0 0 0年《全国病毒性肝炎诊断标准》的慢性肝炎血清 1343份 ,分别用ELISA法、PCR ELISA法检测HB血清标志、HBV -DNA和 1896位点变异株。结果显示 :HBsAg阳性血清 10 97份 (81.6 8%、)HBsAg阴性血清 2 4 6份 (81.31% )。在HBsAg阳性血清中 ,HBsAg、HBeAg、抗 HBc(1 3 5 )阳性组 4 0 4份 (30 .l% ) ,HBV -DNA阳性 347份 (85 .89% ) ,DNA阳性值呈递增趋势 (10 4～ 10 6拷贝 /ml,各占 8.6 5 %、33.71%、5 3.6 1% ) ;而HBsAg、抗 HBe阳性组血清 6 0 6份 (45 .12 % ) ,DNA阳性值 10 5拷贝 /ml,占优势 (6 4 .18% )。在HBsAg阴性血清中 ,抗 HBs、抗 HBe、抗 HBc(2 4 5 )阳性组 2 32份 (17.2 7% ) ,DNA阳性占 7.32 % ,DNA阳性值递减由 10 4～ 10 6拷贝 /ml,各占 5 2 .9%、4 1.l%、5 .9%。结论 ,各血清标志模式中的病毒载量为HBsAg阳性组 >HBsAg阴性组 ,阳性组 1 3 5 >1 4 5 >l 5 >2 4 5。但 1 3 5阳性组中 14 %在界值以下 ,1 4 5阳性组中 1896位点自然变异达 78.6 % ,2 4 5阳性组中仍存在DNA+ 血清 ,以上提示在临床判定和治疗时要慎重对待(注 :l=HBsAg,2 =抗 HBs,3=HBeAg ,4 =抗 HBe ,5 =抗 HBc)     

实时荧光定量PCR检测肺泡灌洗液在HIV阴性马尔尼菲篮状菌病诊断中的价值

目的 探讨实时荧光定量PCR(quantitative real-time PCR,qPCR)技术检测HIV阴性支气管肺泡灌洗液(bronchoalveolar lavage fluid, BALF)对马尔尼菲篮状菌病诊断价值。方法 收集2019年9月—2022年6月就诊于广西医科大学第一附属医院的17例HIV阴性有肺部感染症状的TSM患者及33例肺部其他真菌感染患者或非感染患者的BALF及血清标本,分别提取总DNA,运用qPCR技术检测标本中TM菌载量值,并同时进行BALF真菌培养。结果 同一时间采集的17例外周血和支气管肺泡灌洗液中,在外周血样本中qPCR检测阳性仅11.8%(2/17),BALF真菌培养TM阳性患者同份BALF标本qPCR阳性率可达90.0%(9/10);BALF真菌培养TM阴性患者中,其同份标本有2例qPCR检测呈阳性。33例对照组患者的血清及BALF qPCR均为阴性。其中,BALF qPCR阳性样本的Cq值中位数为31.40,范围为20.71～35.59。同份BALF样本中,BALF qPCR技术(Kappa值：0.708)比BALF培养(Kappa值：0....     

丙型肝炎病毒分片段抗体检测蛋白质芯片的制备及临床评价

为了制备丙型肝炎病毒分片段抗体检测蛋白质芯片,并对其临床应用价值进行评价,将基因工程表达的丙型肝炎病毒分片段抗原,点至经特殊处理的玻片上,制成蛋白质芯片.收集来自三家临床单位用于临床验证的905份血清标本.分别用丙肝病毒分片段抗体检测蛋白质芯片、ELISA丙肝病毒抗体检测试剂进行检测.部分样本同时采用进口RIBA抗体检测试剂进行了检测,分别比较蛋白质芯片法与ELISA法以及RIBA试剂的符合率.结果表明：a.905份血清标本,ELISA法检出阳性294份,阴性611份.阳性标本用蛋白质芯片法检测,融合抗原292份显示阳性结果、2份阴性结果,根据蛋白质芯片的核心抗原,以及NS3, NS4,NS5分片段抗原综合判断确定阳性样本288份阳性,阴性样本2份,4份样本结果不确定.ELISA法检出的611份阴性标本用两种蛋白质芯片法检测,检出阴性均为611份.两种蛋白质芯片法与ELISA法的阳性符合率分别为99.3%和98.9%,与ELISA法的阴性符合率均为100%.用RIBA 试剂检测6份ELISA法为阳性,蛋白质芯片法为非阳性的样本,结果均为非阳性.b.290份经 RIBA试剂确认的阳性标本104份,单片段阳性标本66份,阴性标本120份,用蛋白质芯片法检测,检出阳性标本103份,单片段阳性标本61份,阴性标本126份,二者具有很高的符合率（P＞0.01）.丙型肝炎病毒分片段抗体检测蛋白质芯片,检测灵敏度和特异性高于ELISA法,对血清样本的确认程度与进口的RIBA试剂高度一致,具有操作简便,费用低廉的特点,是一种新型、高效的体外诊断试剂.     

Novel evolved immunoglobulin (Ig)-binding molecules enhance the detection of IgM against hepatitis C virus

Detection of specific antibodies against hepatitis C virus (HCV) is the most widely available test for viral diagnosis and monitoring of HCV infections. However, narrowing the serologic window of anti-HCV detection by enhancing anti-HCV IgM detection has remained to be a problem. Herein, we used LD5, a novel evolved immunoglobulin-binding molecule (NEIBM) with a high affinity for IgM, to develop a new anti-HCV enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-labeled LD5 (HRP-LD5) as the conjugated enzyme complex. The HRP-LD5 assay showed detection efficacy that is comparable with two kinds of domestic diagnostic kits and the Abbott 3.0 kit when tested against the national reference panel. Moreover, the HRP-LD5 assay showed a higher detection rate (55.9%, 95% confidence intervals (95% CI) 0.489, 0.629) than that of a domestic diagnostic ELISA kit (Chang Zheng) (53.3%, 95% CI 0.463, 0.603) in 195 hemodialysis patient serum samples. Five serum samples that were positive using the HRP-LD5 assay and negative with the conventional anti-HCV diagnostic ELISA kits were all positive for HCV RNA, and 4 of them had detectable antibodies when tested with the established anti-HCV IgM assay. An IgM confirmation study revealed the IgM reaction nature of these five serum samples. These results demonstrate that HRP-LD5 improved anti-HCV detection by enhancing the detection of anti-HCV IgM, which may have potential value for the early diagnosis and screening of hepatitis C and other infectious diseases.     

Hepatitis C virus-RNA, immunoglobulin M anti-HCV and risk factors in haemodialysis patients

In order to evaluate hepatitis C virus-RNA (HCV-RNA), immunoglobulin M (IgM) anti-HCV and risk factors in haemodialysis patients, 180 subjects (45 HCV negative and 135 HCV positive) were studied. Sex, age, duration of dialysis, number of transfusions and ALT were also considered. HCV-RNA was determined by the Amplicor HCV test, and IgM anti-HCV by the Abbott HCV IgM EIA. These markers were present in 40% and 30.4% of anti-HCV positive subjects. The agreement between the two tests employed was 77%. The results showed a close association between HCV-RNA and IgM anti-HCV with abnormal ALT levels and between HCV-RNA and the number of transfusions. Both of these markers were different when correlated with age and time on dialysis, respectively. Therefore, IgM anti-HCV may also serve as a serological marker of HCV infection and a complementary marker of virus replication.     

Hepatitis C Viremia Patterns in Incident Hepatitis C Infection and One Year Later in 150 Prospectively Tested Persons Who Inject Drugs

Objectives

To assess HCV viremia levels just before, during and one year after anti-HCV seroconversion in people who inject drugs (PWID).

Methods

PWID enrolling into a needle exchange program in Malmö, Sweden, 1997–2005 constituted the source population. Sera were obtained at enrolment and at approximately 3–4 monthly intervals afterwards, and were initially tested for anti-HIV, HBsAg/anti-HBc and anti-HCV and thereafter for markers previously negative. Seroconversion to anti-HCV had occurred during the study period in 186 out of 332 seronegative subjects. In these anti-HCV seroconverters, quantitative HCV RNA PCR was retrospectively performed on frozen sera to determine viremia levels in the last anti-HCV negative, the first anti-HCV positive and in one year follow-up samples.

Results

Among 150 subjects seroconverting to anti-HCV with samples available from all three defined time-points, eight different patterns of viremia were observed. Spontaneous clearance at one year was noted in 48 cases (32%) and was associated with female gender (p = 0.03, CI 0.17–1.00). In 13 cases HCV-RNA was not detected in any study sample. Among 61 subjects with pre-seroconversion viremia, viral load was significantly higher in the pre-seroconversion samples compared to subsequent samples. For the whole group, viral load declined to undetectable levels at seroconversion in 28% of cases (but with recurrent viremia in 15%).

Conclusions

Different patterns of HCV RNA kinetics were observed among PWID with documented seroconversion to anti-HCV. The frequently observed absence of detectable HCV RNA in the first anti-HCV positive sample (irrespective of subsequent viremia) demonstrates the importance of repeated sampling and RNA testing for determination of the outcome of acute infection.     

Prevalence of low positive anti-HCV antibodies in blood donors: Schistosoma mansoni co-infection and possible role of autoantibodies

Patients infected with schistosoma frequently show a high seroprevalence of anti-hepatitis C virus (anti-HCV) antibodies. The aim of this study was to find the underlying reason for this phenomenon, and to examine a possible involvement of autoantibodies. Out of 2,400 Egyptian blood donors, 192 (8%) were anti-HCV positive by ELISA. They were 133 males and 59 females with age ranging from 27 to 48 years. According to optical density ratio (ODR) of anti-HCV antibodies, 96 cases were low positive (LP) with ODR (1-2) designated as group I, and 96 were high positive (HP) with ODR (> or =2) (group II). Both groups were examined for quantitative HCV core antigen (HCVcAg), liver function (Albumin, ALT, AST) and anti-Schistosoma mansoni(anti-Sm) IgG. Group I cases were HCVcAg negative with normal liver function tests, and 44 of them were anti-Sm positive. Ninety cases (93.75%) of group II were HCVcAg positive with markedly affected liver function tests and 72 cases were anti-Sm positive. All group I cases were examined for autoimmune markers (ANA, AMA, SMA and LKM). In group I, 33 (75%) of anti-Sm positive cases were positive for one or more of the autoimmune markers examined, while none of anti-Sm negative was positive for any marker with significant difference between the two groups (P < 0.0001). Our results primarily on blood donors indicate that LP anti-HCV frequently represents false-positive reactivity with a possible role of Sm-induced autoantibodies in this phenomenon.     

Genetic heterogeneity of hepatitis C virus (HCV) in clinical strains of HIV positive and HIV negative patients chronically infected with HCV genotype 3a

The clinical correlation between the degree of HCV variability and the response to anti-HCV treatment in HIV positive patients infected with HCV genotype 3a is unknown. In this study, 27 HIV positive and 5 HIV negative patients with HCV genotype 3a infection were treated with interferon-alpha-2b with or without ribavirin. Nine patients (5 HIV positive) achieved a sustained virological response (SR) and 23 (only one HIV negative) were non-responders (NR). Sequence analyses of the partial E2 domain and the non-structural 5A protein were performed at baseline in all patients, and before and during treatment in the HIV positive NRs. There was no difference in the mean number of amino acid mutations from HCV 3a prototype, within E2 region, between the HIV positive and HIV negative patients: 17 (range 11-25) vs 16 (range 14-17). The mean baseline number of mutations in E2 region, was similar in HIV positive SRs and NRs: 18 (range 14-25) vs 16 (range 11-19). Phylogenetic analysis of HCV paired serum samples at baseline and during treatment revealed identical E2 sequence in 5/21 HIV positive NR patients, whereas 6 other sequences were strictly related to baseline E2 domain and the remaining 10 were divergent. The mean number of amino acid mutations in the NS5A protein at baseline, was 1 (range 0-3) in HIV negative patients and 2 (range 0-4) in HIV positive ones. This region was highly conserved in all isolates of HIV positive NRs analysed during treatment. These results suggest that genetic variability at baseline within the E2 region and NS5A protein of HCV 3a strain obtained from HIV positive and HIV negative patients is not associated with treatment response. Furthermore, the anti-HCV treatment did not influence HCV heterogeneity within the E2 and NS5A domains in HIV positive patients infected with HCV genotype 3a.     

Determining immunoassay cutoff value using Western blot results to predict hepatitis C infection in blood donors with low-titer anti-HCV reactivity

Since the 1990s, blood donors have been scanned for anti-hepatitis C virus (anti-HCV) antibodies, which can be defined by enzyme immunoassay as a screening test. In this population, false-reactive ratios have been high. Recently, some authors have aimed to find a cutoff value for anti-HCV different from those established by test manufacturers to predict HCV infection. In this study, 321 patients, after two repeating tests, had reactive results in s/co <10 titers on anti-HCV test. The patients were 29.6 % (n?=?95) in women and 70.4 % (n?=?226) in men. The patients were classified into three groups by Western blot (WB) results (PS, positive; NG, negative; and ID, indeterminate). The average anti-HCV titer of the whole group was 2.61?±?1.96. Anti-HCV titers of subgroups were 2.43?±?1.95 in NG, 4.93?±?2.53 in PS, and 2.50?±?1.65 in ID (p?<?0.001). There was a significant difference between NG and PS and between PS and ID subgroups (p?<?0.001). There was a positive correlation between WB and anti-HCV titers in all patients (r?=?0.298, p?<?0.001), in women (r?=?0.282, p?<?0.001), and in men (r?=?0.337, p?=?0.002). According to receiver operator characteristic curve analysis, the cutoff value of anti-HCV titer to predict hepatitis C infection was >2.61 s/co, with 74.1 % sensitivity and 71.6 % specificity (area under the curve, 0.820; 95 % confidence interval, 0.753 to 0.887). We suggest that an effective cutoff value for anti-HCV other than that established by the manufacturer cannot be assigned to predict hepatitis C infection for blood donors in low-prevalence areas.     

Hepatitis B and C in the hemodialysis unit of Tocantins,Brazil: serological and molecular profiles

A survey was conducted in the hemodialysis population of the state of Tocantins, Brazil, aiming to assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, to analyze associated risk factors, and also to investigate these viruses genotypes distribution. During January and March 2001, all patients (n = 100) were interviewed at the unique dialysis unit in Tocantins. Blood samples were collected and serum samples were screened for HBV serological markers. Hepatitis B surface antigen positive samples were tested for HBV DNA. All samples were also tested for anti-HCV antibodies and HCV RNA. An overall prevalence of 45% was found for HBV infection (4% were HBsAg/anti-HBc positive, 2% were anti-HBc only and 39% had anti-HBc/anti-HBs markers). Concerning HCV infection, anti-HCV and HCV RNA were detected in 13% and 14% of the subjects, respectively. Three patients were HCV RNA positive and anti-HCV negative, resulting in an overall HCV prevalence of 16%. Univariate analysis of risk factors showed that only shift and length of tile on hemodialysis were associated with HBV and HCV positivity respectively. Among the four HBsAg-positive samples, HBV DNA was detected in three of them, which were identified as genotype A by restriction fragment length polymorphism (RFLP) analysis. All 14HCV RNA-positive samples were genotyped by INNO-LiPA. Genotypes la and 3a were found in 85% and 15%, respectively. The present data show low HBsAg and HCV prevalence rates. The risk factors associated with HBV and HCV positivity suggest that nosocomial transmission may influence in spreading these viruses in the dialysis unit studied.     

乙型肝炎病人重叠感染丙型肝炎的评价

应用ＥＬＩＳＡ和ＰＣＲ法检测５０２例乙肝病人血清,４０１例ＨＢｓＡｇ阳性血清中,有１１４例（２８．４％）抗－ＨＣＶ和ＨＣＶＲＮＡ双项阳性,２５例（６．２％）ＨＣＶＲＮＡ单项阳性;２１例（５．２％）抗－ＨＣＶ单项阳性。将ＨＢｓＡｇ乙肝病人分成ＨＢＶＤＮＡ,ＨＢｅＡｇ阳性组和ＨＢＶＤＮＡ,ＨＢｅＡｇ阴性组。前者抗－ＨＣＶ阳性率为１１．６％～２０．５％,ＨＣＶＲＮＡ阳性率为１６．２％～２０．５％。后者抗－ＨＣＶ阳性率为２０．２％～５５．６％,ＨＣＶＲＮＡ阳性率为２３％～６０．３％。结果说明长期携带ＨＢＶ者和慢性乙肝病人均可重叠ＨＣＶ感染。ＨＢＶＤＮＡ阳性组抗－ＨＣＶ和ＨＣＶＲＮＡ阳性率明显高于ＨＢＶＤＮＡ阳性组     

Behavior of Medaka fish under distributed gravity.

The threshold value of gravity for Medaka fish (Oryzias latipes) was determined using parabolic flights of an airplane. Rotating a turntable during a 20 sec of microgravity, a gradient field of centrifugal force was realized in the aquarium. Fish of HO5 strain were used because from the previous studies, in microgravity they were known to exhibit looping behavior more easily than any other strains. Looping fish became stable (i.e., recovered their posture control) when fish swam from a lower-gravity area of the aquarium to an area of a certain gravity value or beyond. On the other hand, stable fish lost their posture control and started looping when fish swam into an area of a gravity lower than a certain value. Using these phenomena, we obtained the gravity value of 0.21 to 0.26 G as for the threshold value for Medaka fish to sense the gravity.